Genetic Relationships and Reproductive-isolation Mechanisms among the
Fejervarya limnocharis Complex from Indonesia (Java) and Other Asian
Countries
Author(s) :Tjong Hon Djong, Mohammed Mafizul Islam, Midori Nishioka, Masafumi Matsui,
Hidetoshi Ota, Mitsuru Kuramoto, MdMukhlesur Rahman Khan, Mohammad Shafiqul Alam, De
Silva Anslem, Wichase Khonsue, and Masayuki Sumida
Source: Zoological Science, 24(4):360-375. 2007.
Published By: Zoological Society of Japan
DOI:
URL: http://www.bioone.org/doi/full/10.2108/zsj.24.360
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ZOOLOGICAL SCIENCE 24: 360–375 (2007)
© 2007 Zoological Society of Japan
Genetic Relationships and Reproductive-isolation Mechanisms
among the Fejervarya limnocharis Complex from
Indonesia (Java) and Other Asian Countries
Tjong Hon Djong1,8, Mohammed Mafizul Islam1, Midori Nishioka1, Masafumi Matsui2,
Hidetoshi Ota3, Mitsuru Kuramoto4, Md. Mukhlesur Rahman Khan5,
Mohammad Shafiqul Alam1, De Silva Anslem6, Wichase Khonsue7
and Masayuki Sumida1*
1
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima
739-8526, Japan
2
Graduate School of Human and Environmental Studies, Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan
3
Tropical Biosphere Research Center, University of the Ryukyus,
Nishihara, Okinawa 903-0213, Japan
4
3-6-15 Hikarigaoka, Munakata, Fukuoka 811-3403, Japan
5
Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
6
Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka
7
Department of Biology, Faculty of Science, Chulalongkorn University,
Bangkok 10330, Thailand
8
Department of Biology, Faculty of Science, Andalas University,
Padang 25136, West Sumatra, Indonesia
In order to elucidate the genetic relationships and reproductive-isolation mechanisms among the
Fejervarya limnocharis complex from Indonesia and other Asian countries, allozyme analyses and
crossing experiments were carried out using 208 individuals from 21 localities in eight Asian countries. The allozyme analyses revealed that 17 enzymes examined were controlled by genes at 27
loci, and that 7.9 phenotypes were produced by 5.2 alleles on average. The two species recognized
in F. limnocharis sensu lato from Southeast Asia (i.e., F. limnocharis sensu stricto and F. iskandari)
were found to occur sympatrically at three localities (Bogor, Cianjur and Malingping), all on Java,
Indonesia. Fejervaya iskandari was dominant at each of these localities and showed substantial
geographic genetic variation. Laboratory-produced hybrids between F. limnocharis and F. iskandari
from Java became underdeveloped and died at the tadpole stage, suggesting that these species
are completely isolated by hybrid inviability. Hybrids between topotypic F. limnocharis and the
Malaysian and Japanese conspecific populations developed normally to metamorphosis. Likewise,
hybrids between topotypic F. iskandari and the Thailand and Bangladesh conspecific populations
also showed normal viability throughout larval development. The present allozyme analyses and
crossing experiments strongly suggested the presence of two distinct forms, the large type and the
small type, in the F. limnocharis complex from Asia, and further subdivision of the large type into
the F. limnocharis assemblage and the F. iskandari assemblage. The small type was found in samples from India, Thailand, Bangladesh and Sri Lanka, and included at least three different species.
The sample from Pilok, Thailand, was considered to represent an undescribed species.
Key words: reproductive isolation, Fejervarya limnocharis, Asia, Fejervarya iskandari, genetic relationship
INTRODUCTION
Fejervarya limnocharis sensu lato, known as Rana
limnocharis or Limnonectes limnocharis until recently (Dubois
* Corresponding author. Phone: +81-82-424-7482;
Fax : +81-82-424-0739;
E-mail : [email protected]
doi:10.2108/zsj.24.360
and Ohler, 2000), is a common small-sized frog that occurs
in and around lowland freshwater habitats including paddy
fields. This species is often regarded as one of the most
widely distributed frogs in Asia, from Pakistan to Japan
through Indonesia and other Southeast Asian countries
(Iskandar, 1998; Iskandar and Colijn, 2000; Dubois and
Ohler, 2000). A number of different names have been
proposed for some populations once assigned to F.
Speciation in Asian Fejervarya Complex
limnocharis, but most of these names were immediately
synonymized to F. limnocharis or were used for a long time
but only as subspecific names for this species. This situation
is partly due to the presence of more-or-less recognizable
morphological variations of F. limnocharis sensu lato, and
partly to the absence of diagnostic features to clearly discriminate certain local populations from other populations
(Dubois, 1984, 1987; Inger and Voris, 2001).
Applications of analyses involving non-morphological
characters have recently revealed that F. limnocharis sensu
lato (henceforth referred to as the F. limnocharis complex)
is a composite of many different species. For example,
some populations from Nepal and India, formerly assigned
to F. limnocharis, were shown to represent several different
species chiefly on the basis of differences in their mating call
patterns (Dubois, 1975; Dutta, 1997). Moreover, Toda et al.
(1997, 1998a) provided allozyme data that clearly indicated
cryptic taxonomic diversity in the East Asian and Indonesian
populations of F. limnocharis. Subsequently, Veith et al.
(2001) described one of the cryptic species detected by
Toda et al. (1998a) in Java as a new species, Fejervarya
iskandari, using additional allozyme and mitochondrial DNA
sequence data. Although F. iskandari is morphologically
hardly distinguishable from syntopic populations of F. limnocharis sensu stricto, allozyme data reveal a complete
absence of gene flow between the two species (Toda et al.,
1998a; Veith et al., 2001). This finding suggests that many
more cryptic species may be detected in the F. limnocharis
complex, if further comprehensive and systematic surveys
are carried out for this broadly distributed species complex.
Table 1.
361
In the present study, we carried out allozyme analyses
on samples of the F. limnocharis complex from eight Asian
countries, including those from the type localities of F.
limnocharis (Bogor, Java) and F. iskandari (Cianjur, Java),
to elucidate the genetic relationships among the whole F.
limnocharis complex from Asia. We also carried out crossing
experiments for the available samples from five countries,
including those from the type localities of F. limnocharis and
F. iskandari, in order to obtain information regarding the
mechanisms responsible for the complete reproductive isolation of the two species (see above) and other populations.
MATERIALS AND METHODS
Allozyme analyses
A total of 208 individuals of the F. limnocharis complex (92
male, 70 female and 46 immature frogs) from 21 localities in eight
countries were used (Table 1 and Fig. 1). Samples of a congeneric
and closely related species, Fejervarya cancrivora, were added to
the analyses as an outgroup. Based on the snout-vent length (SVL),
the specimens of the F. limnocharis complex from the eight countries could be classified into two groups, the large type and the
small type (in Kruskal Wallis tests of the SVL, the significance level
of P<0.01 for male X2=16.65 and female X2=10.1). The large type,
recognized from 19 localities mainly in Southeast and East Asia,
exhibited SVLs ranging from 35.0–55.0 mm (mean, 41.2±4.1 mm)
for males and 37.0–56.3 mm (mean, 44.8±5.1 mm) for females. In
contrast, the small type, recognized from four localities in South
Asia and Pilok of Kanchanaburi, Thailand, had SVLs ranging from
27.0–31.9 mm (mean, 30.2±2.0 mm) for males and 30.8–43.4 mm
(mean, 36.4±4.7mm) for females (Table 1).
Seventeen enzymes extracted from skeletal muscles were ana-
Sampling localities, numbers of frogs examined, and population abbreviations used
Species
Locality
Country
F. limnocharis Indonesia
complex
F. cancrivora
Detailed locality
West Java, Bogor*
West Java, Cianjur*
West Java, Malingping
Malaysia
Kuala Lumpur, UMC*
Kota Kinabalu
Thailand
Bangkok
Ranong
Pathumthani, Nongsua*
Saraburi, Muaklek
Kanchanaburi, Pilok, large type
Kanchanaburi, Pilok, small type†
Kanchanaburi, Thongphaphum*
India
Mangalore, Bajipe†
Sri Lanka
Bentota†
Bangladesh Mymensingh, BAU small type†
Mymensingh, BAU large type*
Taiwan
Chiai
Taipei
Pingdong
Japan
Hiroshima, Higashihiroshima*
Okinawa, Iriomote Island
Okinawa, Okinawa Island*
Okinawa Tsuken Island
Indonesia
West Java, Bogor
Total
Number of frogs
Total
Male
Mean SVL (mm)
(range length)
Female
Mean SVL (mm)
(range length)
Immature
23
58
60
3
2
4
2
9
2
2
2
1
2
3
5
5
5
1
1
5
5
5
3
3
211
12
30
14
2
2
1
0
2
2
0
1
1
1
2
2
3
5
0
1
1
5
4
1
0
92
42.1 (35.6–47.0)
41.0 (37.1–44.5)
37.7 (35.0–40.3)
43.4 (41.7–45.0)
41.5 (39.9–41.7)
42.0
–
40.0 (37.0–43.0)
38.0 (35.5–40.4)
–
31.9
48.4
28.4
28.9 (27.0–30.8)
31.6 (31.4–31.8)
49.7 (48.7–51.0)
37.6 (36.1–39.5)
–
45.0
39.5
50.4 (46.0–55.0)
38.0 (37.0–39.0)
38.0
–
8
18
18
1
0
3
2
3
0
2
1
0
1
1
3
2
0
1
0
4
0
1
1
0
70
49.0 (46.4–53.8)
45.6 (40.0–55.1)
39.3 (37.0–42.7)
48.4
–
50.3 (48.0–54.1)
47.7 (45.2–50.0)
43.8 (42.8–45.0)
–
52.3 (52.0–52.7)
43.4
–
31.2
30.8
37.7 (37.6–37.8)
55.2 (54.1–56.3)
–
45.6
–
42.2 (41.6–43.1)
–
38.0
52.3
–
3
10
28
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
3
49
UMC, University of Malaya Campus; BAU, Bangladesh Agricultural University Campus.
* Used for crossing experiments.
†
Small type
Population
abbreviation
Bogo(lim), Bogo(isk)
Cian(lim), Cian(isk)
Mali(lim), Mali(isk)
Kual
Kota
Bang
Rano
Path
Sara
Pilo(L)
Pilo(S)
Thon
Indi
SriL
BAU(S)
BAU(L)
Chia
Taip
Ping
Hiro
Irio
Okin
Tsuk
Bogo(can)
362
T. H. Djong et al.
Fig. 1.
Map showing sampling localities for 26 populations of the Fejervarya limnocharis complex from Asian countries.
Table 2. Enzyme, presumptive locus, enzyme commission number (E.C. No.), tissue and buffer system used for
allozyme analysis in the present study
Enzyme
Locus
E.C. No.
Tissue
Buffer system
Aspartate amintrasferase
Aspartate amintrasferase
Adenosine deaminase
Adenylate kinase
Aldolase
Creatine kinase
Fumarase
Fumarase
α-Glycerophospate dehydrogenase
Glucose-6-phosphate isomerase
Isocitrate dehydrogenase
Isocitrate dehydrogenase
Lactate dehydrogenase
Lactate dehydrogenase
Malate dehydrogenase
Malate dehydrogenase
Malic enzyme
Malic enzyme
Mannose-6-phospate isomerase
Peptidase (valyl-leucine)
Peptidase (leucyl-glycyl-glysine)
Peptidase (leucyl-alanine)
Peptidase (leucyl-proline)
6-Phosphogluconate dehydrogenase
Phosphoglucomutase
Superoxide dismutase
Superoxide dismutase
AAT-1
AAT-2
ADA
AK
ALD
CK
FUM-1
FUM-2
α-GDH
GPI
IDH-1
IDH-2
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
PEP-A
PEP-B
PEP-C
PEP-D
6-PGD
PGM
SOD-1
SOD-2
2.6.1.1
2.6.1.1
3.5.4.4
2.7.4.3
4.1.2.13
2.7.3.2
4.2.1.2
4.2.1.2
1.1.1.8
5.3.1.9
1.1.1.42
1.1.1.42
1.1.1.27
1.1.1.27
1.1.1.37
1.1.1.37
1.1.1.40
1.1.1.40
5.3.1.8
3.4.11/13
3.4.11/13
3.4.11/13
3.4.11/13
1.1.1.44
5.4.2.2
1.15.1.1
1.15.1.1
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
Muscle
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
TEB pH 8.0
TEB pH 8.0
TEB pH 8.0
T-C pH 6.0
TEB pH 8.0
T-C pH 7.0
T-C pH 6.0
T-C pH 6.0
T-C pH 6.0
T-C pH 6.0
T-C pH 6.0
T-C pH 7.0
T-C pH 7.0
T-C pH 7.0
TEB pH 8.0
TEB pH 8.0
TEB pH 8.0
TEB pH 8.0
T-C pH 7.0
TEB pH 8.0
TEB pH 8.0
TEB pH 8.0
Speciation in Asian Fejervarya Complex
lyzed by horizontal starch gel electrophoresis following a procedure
that was previously described in detail (Nishioka et al., 1980, 1992).
The enzymes analyzed are listed in Table 2, together with the corresponding presumptive loci, enzyme commission numbers (E.C.
Nos.), tissues and buffer system names. Each enzyme was
detected using the agar–overlay method outlined by Harris and
Hopkinson (1976). Multiple alleles at each locus were designated
alphabetically in the order of fast to slow anodal mobility.
A locus was considered to be polymorphic when each of multiple alleles existed at a frequency of more than 1% at the locus.
The genetic variability in each population was quantified by calculating the mean proportion of heterozygous loci per individual specimen (H), proportion of polymorphic loci per population (P) and
mean number of alleles per locus (A) (Lewontin, 1974; Berg and
Hamrich, 1997). The syntopic species in one locality was detected
based on a diagnostic allele according to Toda et al. (1998a) and
Veith et al. (2001). The genetic distance (D) and genetic identity (I),
defined by Nei (1972), were also calculated to evaluate the genetic
relationships among populations. Estimates of F-statistics (Fis, Fst
and Fit) for groups or multiple populations (Hartl and Clark, 1989)
were carried out for each group. All parameters were analyzed
using Popgene 1.31 software. The D-value matrices from pairwise
comparisons of samples were transferred to PHYLIP 3.65 software
in order to build a neighbor-joining (NJ) tree (Saitou and Nei, 1987).
Bootstrap (BT) values (Felsenstein, 1985) were also calculated with
PHYLIP 3.65, based on the Nei’s (1972) genetic distances with
1,000 replicates.
Crossing experiments
Crossing experiments were carried out by artificial insemination
among 10 large-type populations from 5 countries (Kawamura et al.,
1981) (Table 1). A total of 36 and 19 combinations were carried out
in the first and second experiments, respectively (Tables 7 and 8).
Ovulation was induced by injecting bullfrog pituitaries into the body
cavity of each female. The tadpoles were fed on boiled spinach.
RESULTS
Allozyme analyses
The electrophoretic patterns revealed that the enzymes
examined were controlled by genes at 27 presumptive loci
(Table 3). Four of these loci (MPI, LDH-B, MDH-1 and ADA)
had high numbers of phenotypes and alleles, while five loci
(AAT-2, AAT-1, AK, FUM-2 and SOD-1) had low numbers.
Among the 27 loci, there were 7.9 phenotypes and 5.2 alleles on average (Table 3).
Analysis of the resultant allelic data for the 27 loci
revealed that F. iskandari and F. limnocharis had diagnostic
alleles at 11 loci (AAT-2, FUM-2, LDH-B, MDH-2, ME-2,
MPI, PEP-A, PEP-C, PEP-D, 6PGD and PGM), and were
present in each of three localities, namely the Bogor, Cianjur
and Malingping populations. The allele frequency at each
locus is shown in Table 4. The geographic distributions of
the PGM and LDH-B alleles among 26 populations of the F.
limnocharis complex are shown in Fig. 2. Among the 26
populations, the mean proportion of heterozygous loci per
individual (H) was 7.7% (range, 0–16.7%), the mean proportion of polymorphic loci per population (P) was 21.5%
(range, 0–63.0%) and the mean number of alleles per locus
(A) was 1.23 (range, 1.00–2.00) (Table 5).
The Fst values calculated among the large-type populations of the F. limnocharis complex were 0.766 on average,
ranging from 0.010 for the AK locus to 0.938 for the MDH-2
locus. The Fis values were low, being –0.090 on average,
with a negative value for 18 polymorphic loci and a positive
363
Table 3. Numbers of alleles and phenotypes at 27 loci.
See Table 2 for abbreviations of loci
Locus
AAT-1
AAT-2
ADA
AK
ALD
CK
FUM-1
FUM-2
α-GDH
GPI
IDH-1
IDH-2
LDH-A
LDH-B
MDH-1
MDH-2
ME-1
ME-2
MPI
PEP-A
PEP-B
PEP-C
PEP-D
6-PGD
PGM
SOD-1
SOD-2
Average
No. of phenotypes
3
2
17
3
5
4
5
3
6
6
4
8
5
20
18
5
10
7
23
7
6
7
9
11
8
3
7
7.9
No. of alleles
3
2
10
3
3
3
4
3
4
4
3
7
3
10
12
4
7
4
14
4
4
6
5
7
5
3
4
5.2
value for 6. The mean Fit value was 0.745. The Fst values
among the small-type populations were 0.843 on average,
ranging from 0.130 for the AAT-1 locus to 0.941 for the ME-1
locus. The Fis values were low, –0.085 on average, with a
negative value for 10 polymorphic loci and a positive value
for 3. The mean Fit value was 0.830.
The genetic distances (D) and genetic identities (I)
among 26 populations of the F. limnocharis complex are
shown in Table 6. The D values were 0.846–1.694 between
the F. limnocharis complex and the outgroup F. cancrivora.
Furthermore, the D values were 0.628–0.749 between two
sympatric species, F. limnocharis and F. iskandari, from
Java; 0.012–0.032 among three populations of F. iskandari
from Java; 0.470–0.614 between F. iskandari from Java and
the Malaysia populations; 0.285–0.431 between F. iskandari
from Java and the Thailand populations; 0.249–0.291
between F. iskandari from Java and the Bangladesh populations; and 0.300–0.455 between F. iskandari from Java
and the Taiwanese-Japanese populations. The distances
were 0.046–0.076 among three populations of F. limnocharis from Java; 0.410–0.526 between F. limnocharis from
Java and the Malaysia populations; 0.250–0.572 between F.
limnocharis from Java and the Thailand populations; 0.509–
0.579 between F. limnocharis from Java and the Bangladesh populations; and 0.365–0.638 between F. limnocharis from Java and the Taiwanese-Japanese populations.
The D values among six populations from Thailand were
0.056–0.197, while those among seven populations from
Taiwan and Japan were 0.027–0.290. The genetic distances
between the small-type populations, including those from
364
T. H. Djong et al.
Table 4. Allele frequencies at 27 loci in 26 Asian populations of the F. limnocahris complex. See Table 1 and Table 2 for abbreviations for
populations and loci, respectively
Locus
AAT-1
AAT-2
ADA
AK
Bogo (isk) Cian (isk)
b (1.000) a (0.947)
b (0.053)
b (1.000) b (1.000)
c (0.088) d (0.061)
d (0.088) f (0.938)
f (0.794)
h (0.029)
c (1.000) c (1.000)
Bogo (lim)
b (1.000)
Cian (lim)
b (1.000)
Mali (lim)
b (1.000)
a (1.000)
h (0.917)
i (0.083)
a (1.000)
h (1.000)
a (1.000)
g (0.125)
h (0.708)
i (0.167)
a (0.010)
b (0.990)
b (0.854)
c (0.146)
c (1.000)
c (1.000)
c (1.000)
b (0.093)
c (0.917)
c (1.000)
b (1.000)
b (1.000)
a (0.042)
b (0.958)
c (1.000)
c (1.000)
Kota
a (0.250)
b (0.750)
a (1.000)
i (1.000)
Rano
a (0.250)
b (0.750)
a (1.000)
d (0.250)
f (0.250)
h (0.500)
c (1.000)
c (1.000) C (1.000) c (1.000)
c (1.000)
b (1.000)
b (1.000)
b (1.000) b (1.000)
b (1.000)
b (1.000) b (1.000)
a (0.750)
b (0.250)
b (1.000)
b (1.000)
b (1.000)
b (1.000) b (1.000)
b (1.000)
b (1.000) b (1.000)
b (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
a (1.000)
c (1.000)
a (1.000)
c (1.000)
a (1.000)
c (1.000)
a (1.000)
c (1.000)
b (0.990)
c (0.010)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
a (1.000) a (1.000) a (1.000)
b (0.333) c (0.875)) c (0.750)
c (0.667) d (0.125) d (0.250)
c (1.000) c (1.000) b (1.000)
a (1.000)
c (0.750)
d (0.250)
c (1.000)
a (1.000)
b (0.250)
c (0.750)
c (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000) b (1.000)
b (1.000)
b (1.000)
b (1.000)
e (0.010)
f (0.906)
g (0.083)
c (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
a (0.167)
b (0.833)
d (0.083)
f (0.917)
b (1.000)
b (1.000)
a (1.000)
a (1.000) b (1.000)
b (1.000)
b (0.167)
c (0.833)
a (1.000)
b (0.056)
c (0.944)
b (0.889)
c (0.111)
a (0.056)
b (0.833)
c (0.111)
a (0.056)
f (0.888)
g (0.056)
b (1.000)
b (1.000)
f (1.000)
e (0.458)
f (0.500)
g (0.042)
g (1.000)
g (1.000)
c (0.125)
e (0.500)
f (0.125)
j (0.250)
d (1.000) d (0.250)
f (0.125)
h (0.625)
e (1.000)
b (0.750)
c (0.250)
e (0.722) e (1.000)
g (0.111)
j (0.167)
d (1.000)
a (1.000)
c (1.000)
c (1.000)
b (0.056) f (0.750)
d (0.056) g (0.250)
e (0.278)
f (0.167)
h (0.444)
c (1.000) c (1.000)
e (1.000)
e(0.944) c (0.250)
g (0.056) e (0.750)
a (0.167) b (1.000)
b (0.833)
e (1.000)
g (0.111) I (0.250)
h (0.333) j (0.750)
i (0.222)
k (0.278)
l (0.056)
a (0.167) b (0.750)
b (0.778) c (0.250)
c (0.056)
c (1.000) c (1.000)
g (0.750)
k (0.250)
b (0.056)
c (0.944)
c (0.833)
d (0.167)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (0.056) d (1.000)
d (0.944)
c (0.722) c (1.000)
d (0.278)
a (1.000) a (1.000)
d (1.000) d (1.000)
d (1.000)
b (0.970)
c (0.030)
CK
FUM-1
a (0.030)
b (0.970)
c (1.000)
FUM-2
α-GDH
c (1.000)
c (1.000)
a (0.035)
b (0.965)
c (1.000)
c (1.000)
GPI
b (1.000)
b (1.000)
IDH-1
b (1.000)
b (1.000)
IDH-2
f (1.000)
f (1.000)
LDH-A
b (0.088)
c (0.912)
a (0.029)
b (0.765)
c (0.206)
c (1.000)
a (0.105)
b (0.456)
c (0.439)
b (0.490)
c (0.510)
MDH-1
d (0.412)
f (0.176)
h (0.323)
i (0.083)
d (0.675)
f (0.018)
h (0.289)
I (0.018)
b (0.125)
d (0.677)
h (0.198)
d (0.833)
f (0.167)
d (1.000)
d (1.000)
d (1.000)
MDH-2
c (1.000)
c (1.000)
c (1.000)
a (1.000)
a (1.000)
a (1.000)
ME-1
e (1.000)
e (1.000)
e (1.000)
b (0.942)
c (0.029)
d (0.029)
h (0.676)
j (0.323)
c (0.010)
e (0.990)
b (0.958)
d (0.042)
e (1.000)
ME-2
e (0.947)
f (0.053)
b (0.991)
d (0.009)
a (0.750)
b (0.250)
e (1.000)
b (0.083)
d (0.917)
d (1.000)
d (1.000)
b (1.000)
e (0.333) e (0.750)
g (0.667) g (0.250)
b (1.000) b (1.000)
g (0.017)
h (0.597)
j (0.386)
h (0.302)
j (0.698)
i (0.083)
k (0.834)
l (0.083)
i (1.000)
i (0.250)
k (0.750)
j (1.000)
j (1.000)
h (0.125)
I (0.125)
k (0.625)
l (0.125)
i (0.500)
k (0.250)
l (0.250)
MPI
c (1.000)
d (0.500)
h (0.500)
b (0.750)
d (0.250)
PEP-A
b (1.000)
b (1.000)
b (1.000)
c (0.750)
d (0.250)
c (1.000)
c (0.875)
d (0.125)
b (1.000)
b (1.000) b (1.000)
b (1.000)
PEP-B
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
b (1.000)
b (0.974)
c (0.026)
b (1.000)
c (1.000)
c (1.000)
b (0.333)
c (0.667)
d (1.000)
c (1.000)
b (1.000)
b (0.250)
c (1.000)
d (1.000)
c (1.000)
PEP-D
a (0.083)
b (0.917)
c (0.750)
d (0.250)
b (0.333)
c (0.667)
b (1.000)
c (1.000)
c (1.000)
c (0.375)
d (0.625)
c (1.000)
c (1.000)
PEP-C
c (0.991)
d (0.009)
c (1.000)
c (1.000)
c (1.000)
6PGD
d (1.000)
c (1.000)
c (1.000)
d (1.000)
c (1.000)
b (1.000)
b (1.000)
b (1.000)
b (0.250)
c (0.750)
c (1.000)
c (1.000)
a (1.000)
SOD-1
SOD-2
a (1.000)
c (0.029)
d (0.971)
a (1.000)
c (0.035)
d (0.917)
d (0.990)
e (0.010)
a (0.958)
b (0.042)
a (1.000)
c (0.010)
d (0.990)
c (1.000)
PGM
d (0.895)
e (0.105)
a (1.000)
a (1.000)
d (1.000)
a (1.000)
d (1.000)
a (1.000)
c (0.083)
d (0.917)
b (0.833)
c (0.167)
a (1.000)
d (1.000)
c (0.875)) c (1.000)
d (0.125)
a (1.000) a (1.000)
d (1.000) d (1.000)
a (1.000)
d (1.000)
Path
Sara
a (0.056) b (1.000)
b (0.944)
a (1.000) a (1.000)
f (0.333) f (0.250)
h (0.667) h (0.750)
Population
Pilo (L)
b (1.000)
Kual
Bang
a (0.167) b (1.000)
b (0.833)
a (1.000) a (1.000)
h (1.000) f (0.125)
h (0.625
I (0.125)
j (0.125)
c (1.000) c (1.000)
ALD
LDH-B
a (0.018)
b (0.614)
c (0.368)
b (1.000)
Mali (isk)
a (0.990)
b (0.010)
b (1.000)
d (0.073)
f (0.667)
g (0.260)
a (1.000)
f (1.000)
e (0.250)
j (0.750)
c (1.000)
b (1.000)
b (1.000)
c (1.000)
c (1.000)
a (1.000)
d (1.000)
Speciation in Asian Fejervarya Complex
Thon
b (1.000)
Pilo (S)
b (1.000)
Indi
b (1.000)
SriL
b (1.000)
a (1.000)
f (1.000)
b (1.000)
f (0.5.00)
h (0.500)
b (1.000)
e (1.000)
b (1.000)
c (1.000)
c (1.000)
a (1.000)
c (1.000)
b (1.000)
c (1.000)
b (1.000)
c (1.000)
365
BAU (S)
a (0.100)
b (0.900)
b (1.000)
b (0.100)
c (0.900)
BAU (L)
a (0.100)
b (0.900)
a (1.000)
h (1.000)
Chia
b (1.000)
Taip
b (1.000)
Ping
b (1.000)
Hiro
Irio
b (1.000) b (1.000)
Okin
b (1.000)
Tsuk
b (1.000)
Bogo(can)
b (1.000)
a (1.000)
h (1.000)
a (1.000)
h (1.000)
a (1.000)
h (1.000)
a (1.000) a (1.000)
h (1.000) h (1.000)
a (1.000)
h (1.000)
a (1.000)
h (1.000)
a (1.000)
a (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
a (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000) b (1.000)
b (1.000)
b (1.000)
b (1.000)
c (1.000)
a (1.000)
a (1.000)
a (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000) b (1.000)
b (1.000)
b (1.000)
b (1.000)
c (1.000)
c (1.000)
d (1.000)
d (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
d (1.000)
a (1.000) B (1.000) a (1.000)
b (0.500) b (0.500) a (1.000)
c (0.500) c (0.500)
c (1.000) b (1.000) a (0.750)
b (0.250)
b (1.000) b (1.000) b (1.000)
a (1.000)
a (1.000)
a (1.000)
a (0.900)
c (0.100)
a (1.000)
c (1.000)
b (0.100)
c (0.900)
b (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
b (0.100)
c (0.900)
b (1.000) c (1.000)
c (1.000)
c (1.000)
b (0.833)
c (0.167)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (0.400) b (1.000)
c (0.600)
c (0.900)
d (0.100)
b (1.000)
c (1.000)
b (0.333)
c (0.667)
c (0.667)
d (0.333)
b (1.000)
c (1.000)
d (1.000)
c (1.000)
c (1.000)
b (0.500)
c (0.500)
c (1.000)
f (1.000)
e (1.000)
c (0.500)
f (0.500)
b (0.167)
c (0.833)
c (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
d (1.000)
b (1.000)
c (1.000)
b (1.000)
b (1.000)
b (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
b (1.000)
e (1.000)
c (1.000)
a (1.000)
a (0.333)
h (0.667)
d (1.000)
b (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (1.000)
f (0.700)
I (0.300)
f (1.000)
h (1.000)
j (0.100)
k (0.900)
d (0.100)
h (0.900)
a (0.100)
c (0.700)
e (0.200)
c(1.000)
c(1.000)
c (1.000)
a (0.100)
c (0.900)
a (0.200)
c (0.800)
a (0.167)
c (0.853)
f (1.000)
d (0.250)
h (0.750)
k (0.750)
l (0.250)
k (1.000)
f (1.000)
c (1.000)
c (1.000)
d (1.000)
d (1.000)
d (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
a (1.000)
c (1.000)
c (1.000)
c (1.000)
e (1.000)
e (1.000)
b (1.000)
a (1.000)
e (1.000)
e (1.000)
e (1.000)
e (1.000)
e (1.000)
e (1.000)
c (1.000)
b (1.000)
b (1.000)
c (1.000)
c (1.000)
d (0.100)
f (0.900)
c (1.000)
c (0.900)
d (0.100)
e (1.000) e (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
c (1.000)
b (1.000)
b (1.000)
a (1.000)
i (1.000)
f (0.500)
l (0.250)
n (0.250)
a (0.250)
b (0.500)
c (0.250)
b (0.167) b (1.000)
e (0.167)
i (0.667)
d (1.000)
g (1.000)
g (1.000)
g (1.000)
g (1.000)
i (0.800) g (1.000)
m (0.200)
g (1.000)
f (0.500)
l (0.500)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
b (1.000)
a (0.100) b (1.000)
b (0.900)
a (0.100)
b (0.900)
b (0.667)
c (0.333)
b (0.667)
c (0.333)
c (1.000)
a (1.000)
a (1.000)
b (1.000)
b (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
d (1.000)
c (1.000)
d (1.000)
d (1.000)
c (1.000)
e (1.000)
c (1.000)
c (1.000)
f (1.000)
e (1.000)
a (1.000)
a (0.900)
b (0.100)
b (0.200)
c (0.800)
c (1.000)
d (1.000)
d (1.000)
e (1.000)
f (1.000)
a (1.000)
d (1.000)
c (1.000)
d (1.000)
c (0.833)
d (0.167)
c (1.000)
g (1.000)
c (1.000)
b (0.400)
f (0.600)
d (1.000)
c (0.100)
d (0.800)
e (0.100)
b (0.100)
c (0.900)
c (1.000)
b (0.300) b (1.000)
c (0.700)
b (0.300) c (0.100)
c (0.700) d (0.900)
b (1.000)
e (1.000)
b (0.500)
c (0.500)
c (1.000)
b (1.000)
c (0.500)
d (0.500)
b (0.100)
c (0.900)
c (1.000)
a (1.000)
d (1.000)
c (1.000)
a (1.000)
b (1.000)
b (0.750)
d (0.250)
c (1.000)
a (1.000)
c (1.000)
a (1.000)
a (1.000)
d (1.000)
a (1.000)
d (1.000)
a (1.000)
c (1.000)
a (1.000)
b (0.500)
c (0.500)
c (1.000)
c (1.000)
d (1.000)
d (1.000)
c (1.000)
d (1.000)
c (1.000)
c (1.000)
c (1.000)
c (1.000)
a (1.000)
d (1.000)
a (1.000)
d (1.000)
a (1.000)
d (1.000)
c (0.600) c (1.000)
d (0.400)
a (1.000) a (1.000)
d (1.000) d (1.000)
c (0.500)
d (0.500)
a (1.000)
d (1.000)
c (1.000)
b (1.000)
e (1.000)
366
T. H. Djong et al.
Fig. 2. Geographic distributions of the (A) PGM and (B) LDH-B alleles among 26 Asian populations of the Fejervarya limnocharis complex.
See Table 1 for population abbreviations.
Speciation in Asian Fejervarya Complex
were 0.181–1.310.
The NJ tree based on genetic distances revealed that
the F. limnocharis complex diverged into two groups, the
large-type and small-type groups, with BT values of 100%
the Pilok (Kanchanaburi, Thailand), Mangalore (India), Bentota (Sri Lanka) and Mymensingh (BAU(S), Bangladesh)
populations, and the large-type populations were 0.709–
1.730, while the D values among the small-type populations
Table 5.
Species
367
Genetic variabilities at 27 loci in 26 Asian populations of the F. limnocharis complex
Country, Locality
Population
abbreviation
F. iskandari
Indonesia, Bogor
Indonesia, Cianjur
Indonesia, Malingping
F. limnocharis Indonesia, Bogor
complex
Indonesia, Cianjur
Indonesia, Malingping
Malaysia, Kota Kinabalu
Malaysia, Kuala Lumpur
Thailand, Bangkok
Thailand, Ranong
Thailand, Pathumthani
Thailand, Saraburi
Thailand, Pilok
Thailand, Thongphaphum
Thailand, Pilok
India, Mangalore
Sri Lanka, Bentota
Bangladesh, Mymensingh
Bangladesh, Mymensingh
Taiwan, Chiai
Taiwan, Taipei
Taiwan, Pingdong
Japan, Higashihiroshima
Japan, Iriomote
Japan, Okinawa
Japan, Tsuken
Average
Sample
size
Bogo (isk)
Cian (isk)
Mali (isk)
Bogo (lim)
Cian (lim)
Mali (lim)
Kota
Kual
Bang
Rano
Path
Sara
Pilo (L)
Thon
Pilo (S)
Indi
SriL
BAU (S)
BAU (L)
Chia
Taip
Ping
Hiro
Irio
Okin
Tsuk
Mean proportion of heterozygous
loci per individual (%)
17
57
48
6
1
12
2
3
4
2
9
2
2
1
2
2
3
5
5
5
1
1
5
5
5
3
8.0
Proportion of polymorphic
loci per population (%)
8.1 ( 8.7)
9.8 (10.2)
11.0 (11.4)
9.9 ( 9.2)
0 ( 0 )
9.0 ( 7.5)
7.4 ( 5.6)
9.9 ( 7.0)
13.0 (11.9)
16.7 (10.7)
15.2 (19.0)
13.0 ( 9.7)
7.4 ( 5.5)
7.4 ( 3.7)
9.3 ( 7.4)
7.4 ( 8.3)
6.2 ( 4.9)
5.9 ( 5.8)
3.7 ( 4.4)
3.0 ( 2.4)
7.4 ( 3.7)
3.7 ( 1.9)
6.7 ( 7.3)
4.4 ( 3.9)
5.9 ( 7.1)
9.9 ( 7.0)
7.7 ( 6.8)
Mean number of
alleles per locus
33.3
48.2
59.3
37.0
0
22.2
14.8
18.5
25.9
22.2
63.0
25.9
14.8
7.4
14.8
18.5
14.8
25.9
18.5
7.4
7.4
3.7
18.5
18.5
22.2
18.5
21.5
1.55
1.67
1.70
1.41
1.00
1.30
1.15
1.18
1.52
1.30
2.00
1.26
1.15
1.07
1.18
1.22
1.18
1.26
1.22
1.11
1.07
1.04
1.18
1.18
1.22
1.19
1.23
Parentheses show expected values.
Tabel 6. Nei’s (1972) genetic distance (below diagonal) and genetic identity (above diagonal) among 26 Asian populations of the Fejervarya
limnocahris complex. See Table 1 for population abbreviations
Population
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1. Bogo (isk)
2. Cian (isk)
3. Mali (isk)
4. Bogo (lim)
5. Cian (lim)
6. Mali (lim)
7. Kota
8. Kual
9. Bang
10. Rano
11. Path
12. Sara
13. Pilo (L)
14. Thon
15. Pilo (S)
16. Indi
17. SriL
18. BAU (S)
19. BAU (L)
20. Chia
21. Taip
22. Ping
23. Hiro
24. Irio
25. Okin
26. Tsuk
27. Bogo (can)
–
0.012
0.032
0.664
0.720
0.628
0.470
0.596
0.345
0.370
0.285
0.366
0.377
0.373
0.786
1.107
1.269
1.392
0.249
0.367
0.300
0.336
0.375
0.418
0.388
0.425
1.162
0.988
–
0.027
0.638
0.685
0.646
0.484
0.612
0.370
0.386
0.314
0.394
0.364
0.403
0.709
1.167
1.359
1.495
0.274
0.376
0.314
0.350
0.386
0.442
0.399
0.437
1.238
0.968
0.973
–
0.698
0.749
0.661
0.483
0.614
0.390
0.412
0.340
0.397
0.404
0.431
0.733
1.145
1.313
1.442
0.291
0.393
0.324
0.361
0.404
0.452
0.416
0.455
1.112
0.515
0.528
0.498
–
0.046
0.058
0.437
0.410
0.427
0.323
0.369
0.488
0.480
0.572
1.237
1.270
1.652
1.594
0.514
0.473
0.599
0.516
0.365
0.513
0.423
0.418
1.353
0.487
0.504
0.473
0.956
–
0.076
0.526
0.487
0.476
0.335
0.404
0.504
0.510
0.569
1.276
1.306
1.566
1.637
0.579
0.505
0.638
0.546
0.401
0.516
0.456
0.436
1.432
0.534
0.524
0.516
0.944
0.927
–
0.428
0.417
0.373
0.250
0.310
0.417
0.453
0.500
1.410
1.137
1.413
1.392
0.509
0.469
0.602
0.510
0.365
0.519
0.410
0.402
1.241
0.625
0.616
0.617
0.646
0.591
0.652
–
0.144
0.363
0.287
0.288
0.354
0.435
0.386
1.248
1.055
1.396
1.463
0.311
0.399
0.476
0.470
0.353
0.488
0.449
0.461
1.246
0.551
0.543
0.541
0.664
0.615
0.659
0.866
–
0.347
0.395
0.374
0.337
0.469
0.408
1.402
1.168
1.388
1.466
0.394
0.367
0.437
0.447
0.400
0.455
0.387
0.427
1.102
0.708
0.691
0.677
0.652
0.622
0.689
0.695
0.707
–
0.115
0.056
0.057
0.097
0.103
1.065
0.925
1.122
1.227
0.285
0.217
0.202
0.191
0.382
0.308
0.281
0.281
0.908
0.691
0.680
0.662
0.724
0.715
0.779
0.751
0.674
0.891
–
0.073
0.147
0.197
0.170
1.025
0.919
1.145
1.282
0.220
0.250
0.337
0.326
0.308
0.427
0.315
0.313
1.048
0.752
0.731
0.712
0.691
0.668
0.734
0.750
0.688
0.945
0.930
–
0.088
0.145
0.116
0.989
0.946
1.110
1.226
0.239
0.265
0.255
0.242
0.317
0.344
0.315
0.313
0.981
0.694
0.674
0.673
0.614
0.604
0.659
0.702
0.714
0.945
0.863
0.916
–
0.145
0.074
1.175
0.977
1.205
1.332
0.322
0.230
0.212
0.203
0.399
0.314
0.297
0.282
0.868
0.686
0.695
0.668
0.619
0.600
0.636
0.647
0.626
0.907
0.822
0.865
0.865
–
0.135
1.119
1.027
1.284
1.393
0.408
0.277
0.253
0.250
0.454
0.417
0.348
0.336
1.062
0.689
0.669
0.650
0.564
0.566
0.607
0.680
0.665
0.902
0.844
0.890
0.929
0.874
–
1.159
0.956
1.112
1.301
0.372
0.326
0.275
0.297
0.501
0.329
0.395
0.379
0.846
0.456
0.492
0.480
0.290
0.279
0.244
0.287
0.246
0.345
0.359
0.372
0.309
0.327
0.314
–
1.104
1.310
1.289
0.864
1.048
1.041
1.051
1.056
1.171
0.980
1.062
1.622
0.331
0.311
0.318
0.281
0.271
0.321
0.348
0.311
0.397
0.399
0.388
0.377
0.358
0.384
0.332
–
0.588
0.526
1.150
1.193
1.186
1.195
1.283
1.052
1.248
1.212
1.694
0.281
0.257
0.269
0.192
0.209
0.244
0.248
0.250
0.326
0.318
0.330
0.300
0.277
0.329
0.270
0.556
–
0.181
1.502
1.512
1.488
1.587
1.540
1.384
1.579
1.730
1.407
0.249
0.224
0.237
0.203
0.195
0.249
0.232
0.231
0.293
0.278
0.294
0.264
0.248
0.272
0.276
0.591
0.835
–
1.633
1.625
1.638
1.628
1.666
1.438
1.528
1.675
1.532
0.780
0.760
0.748
0.598
0.561
0.601
0.733
0.674
0.752
0.803
0.788
0.725
0.665
0.689
0.421
0.317
0.223
0.195
–
0.188
0.236
0.248
0.226
0.291
0.237
0.243
1.018
0.693
0.687
0.675
0.623
0.604
0.626
0.671
0.693
0.805
0.779
0.767
0.795
0.758
0.722
0.351
0.303
0.220
0.197
0.828
–
0.062
0.048
0.112
0.190
0.049
0.048
0.997
0.741
0.730
0.724
0.550
0.528
0.548
0.621
0.646
0.817
0.714
0.775
0.809
0.777
0.760
0.353
0.306
0.226
0.194
0.790
0.940
–
0.029
0.166
0.151
0.113
0.091
0.991
0.715
0.705
0.697
0.597
0.580
0.601
0.625
0.640
0.827
0.722
0.785
0.816
0.779
0.743
0.350
0.303
0.205
0.196
0.780
0.954
0.972
–
0.153
0.118
0.067
0.052
1.000
0.687
0.680
0.668
0.695
0.670
0.694
0.703
0.670
0.683
0.735
0.728
0.671
0.635
0.606
0.348
0.277
0.214
0.189
0.797
0.894
0.847
0.858
–
0.290
0.124
0.134
1.294
0.658
0.643
0.636
0.599
0.597
0.595
0.614
0.635
0.735
0.652
0.709
0.731
0.659
0.720
0.310
0.349
0.251
0.237
0.748
0.827
0.860
0.888
0.748
–
0.166
0.153
0.910
0.678
0.671
0.660
0.655
0.634
0.664
0.639
0.679
0.755
0.730
0.730
0.743
0.706
0.674
0.375
0.287
0.206
0.217
0.789
0.952
0.893
0.935
0.884
0.847
–
0.027
0.999
0.654
0.646
0.634
0.658
0.647
0.669
0.631
0.653
0.755
0.731
0.731
0.755
0.715
0.685
0.346
0.298
0.177
0.187
0.784
0.954
0.913
0.950
0.875
0.858
0.974
–
1.020
0.313
0.290
0.329
0.259
0.239
0.289
0.288
0.332
0.403
0.351
0.375
0.420
0.346
0.429
0.198
0.184
0.245
0.216
0.361
0.369
0.371
0.368
0.274
0.402
0.372
0.361
–
368
T. H. Djong et al.
and 72.7%, respectively (Fig. 3). The large-type group was
further divided into two subgroups, of which the first subgroup consisted of the F. iskandari populations from Indonesia, the Bangladesh population and the Taiwanese-Japanese populations, and the second subgroup consisted of the
Thailand populations, the F. limnocharis populations and the
Malaysian populations. The assemblage clades in these
subgroups were not strongly supported, since the BT values
were below 50%. The Thailand population showed a paraphyletic assemblage relationship with the Malaysia and F.
limnocharis populations, and another population showed a
monophyletic assemblage relationship. In the small-type
group, the Thailand (Pilok) population first diverged from the
remainder (BT, 94.2%), followed by the Indian population,
leaving the Sri Lanka and Bangladesh populations monophyletic (BT, 97.3%) (Fig. 3).
Crossing experiments
The developmental capacities of the hybrids among 10
populations from five countries and the controls are shown
in Tables 7 and 8. In the first crossing experiments involving
36 control and hybrid mating combinations of the F.
limnocharis complex, except for combinations using females
from Thailand, the eggs cleaved normally with frequencies
ranging from 43.8–98.5% (x– =81.1%), became normal tailbud embryos with frequencies ranging from 20.5–81.7% (x–
=46.2%), hatched normally with frequencies ranging from
18.1–70.2% (x– =40.6%), became normally feeding tadpoles
with frequencies ranging from 11.2–68.2% (x– =32.7%),
became normal 30-day-old tadpoles with frequencies ranging
from 0–54.5% (x– =16.6%) or underdeveloped with frequencies ranging from 0–55.6% (x– =10.6%), and finally metamorphosed normally with frequencies ranging from 0–49.6% (x–
=16.3%). In the second crossing experiments involving 19
control and hybrid mating combinations of the F. limnocharis
complex, the eggs cleaved normally with frequencies ranging from 34.6–99.2% (x– =89%), became normal tail-bud
embryos with frequencies ranging from 27.6–96.8% (x–
=69.8%), hatched normally with frequencies ranging from
5.3–93.7% (x– =61.8%), became normally feeding tadpoles
with frequencies ranging from 3.5–92.9% (x– =49.5%),
became normal 30-day-old tadpoles with frequencies ranging from 0–78.1% (x– =22.0%), and finally metamorphosed
normally with frequencies ranging from 0–76.0% (x– =25.5%).
Fig. 3. Neighbor-joining tree among 26 Asian populations of the Fejervarya limnocharis complex based on genetic distances as defined by
Nei (1972). The value for each branch shows the bootstrap p-value (>50%) for 1,000 replicates. The scale bar represents branch length in
terms of Nei’s genetic distance.
Speciation in Asian Fejervarya Complex
369
Table 7. Developmental capacity of the hybrid among 7 Asian populations of the F. limnocharis complex and the controls in the first crossing
experiments
Parents
Female
No. of eggs No. of normally
cleaved eggs
Male
No. of normal
No. of normally
No. of normally
No. of 30-day-old tadpoles
tail-bud embryos hatched tadpoles feeding tadpoles
Normal
No. of tadpoles
examined
and chromosome
number
Underdeveloped
(%)
No. of normally
No. of tadpoles
examined
and chromosome
number
metamorphosed
frogs
(%)
(%)
(%)
(%)
(%)
2n
3n
2n
3n
(%)
Hiroshima-1
Hiroshima-1
61
43 (70.5)
43 (70.5)
39 (63.9)
17 (27.9)
16 (26.2)
4
1
0
0
0
16 (26.2)
Okinawa
Hiroshima-1
196
181 (92.4)
99 (50.5)
55 (28.1)
50 (25.5)
51 (26.0)
–
–
0
0
0
50 (25.5)
Malaysia
Hiroshima-1
435
418 (96.1)
132 (30.3)
114 (26.2)
90 (20.7)
84 (19.3)
–
–
4 ( 0.9)
–
–
84 (19.3)
Thailand-1
Hiroshima-1
121
11 ( 9.1)
11 ( 9.1)
11 ( 9.1)
11 ( 9.1)
4 ( 3.3)
0
4
7 ( 5.8)
5
0
1 ( 0.8)
Bangladesh-1 Hiroshima-1
247
182 (73.7)
129 (52.2)
123 (49.8)
117 (47.4)
0
0
0
105 (42.5)
–
–
9 ( 3.6)
Bogor (isk)-1
Hiroshima-1
22
15 (68.2)
8 (36.4)
8 (36.4)
8 (36.4)
1 ( 4.6)
0
1
5 (22.7)
5
0
Hiroshima-1
Bogor (lim)
208
198 (95.2)
170 (81.7)
146 (70.2)
103 (49.5)
95 (45.7)
–
–
7 ( 3.4)
–
–
88 (42.3)
0
Okinawa
Bogor (lim)
299
259 (86.6)
132 (44.2)
122 (40.8)
103 (34.5)
101 (33.8)
–
–
1 ( 0.3)
–
–
102 (34.1)
Malaysia
Bogor (lim)
278
235 (84.5)
102 (36.7)
75 (27.0)
49 (17.6)
46 (16.6)
–
–
2 ( 0.7)
–
–
43 (15.5)
Thailand-1
Bogor (lim)
281
104 (37.0)
104 (37.0)
103 (36.7)
102 (36.3)
0
0
0
100 (35.6)
5
0
85 (30.3)
Bangladesh-1 Bogor (lim)
250
219 (87.6)
70 (28.0)
62 (24.8)
56 (22.4)
0
0
0
48 (19.2)
5
0
11 ( 4.4)
Bogor (isk)-1
Bogor (lim)
126
118 (93.7)
90 (71.4)
87 (69.1)
86 (68.3)
5 ( 4.0)
0
5
70 (55.6)
5
0
6 ( 4.8)
Bogor (isk)-2
Bogor (lim)
89
64 (71.9)
32 (36.0)
19 (21.4)
10 (11.2)
3 ( 3.4)
0
3
7 ( 7.8)
5
0
1 ( 1.1)
Hiroshima-1
Malaysia
196
193 (98.5)
115 (58.7)
97 (49.5)
67 (34.2)
64 (32.7)
–
–
3 ( 1.5)
–
–
63 (32.1)
Okinawa
Malaysia
266
242 (91.0)
167 (62.8)
158 (59.4)
142 (53.4)
138 (51.9)
–
–
4 ( 1.5)
–
–
132 (49.6)
Malaysia
Malaysia
328
289 (88.1)
121 (36.9)
121 (36.9)
82 (25.0)
59 (18.0)
5
0
4 ( 1.2)
–
–
53 (16.2)
Thailand-1
Malaysia
232
114 (49.1)
114 (49.1)
114 (49.1)
110 (47.4)
0
0
0
110 (47.4)
5
0
95 (41.0)
Bangladesh-1 Malaysia
244
211 (86.5)
124 (50.8)
121 (49.6)
82 (33.6)
0
0
0
93 (38.1)
5
0
34 (13.9)
88
83 (94.3)
23 (26.1)
23 (26.1)
19 (21.6)
1 ( 1.1)
0
1
18 (20.5)
5
0
1 ( 1.1)
0
0
0
79 (30.4)
–
–
0
Bogor (isk)-1
Malaysia
Okinawa
Thailand-1
260
245 (94.2)
113 (43.5)
105 (40.4)
95 (36.5)
Malaysia
Thailand-1
281
179 (63.7)
140 (49.8)
132 (47.0)
101 (35.9)
1 ( 0.4)
0
1
48 (17.1)
5
0
Thailand-1
Thailand-1
182
69 (37.9)
69 (37.9)
66 (36.7)
64 (36.2)
49 (26.9)
5
0
5 ( 2.8)
–
–
40 (22.0)
Bangladesh-1 Thailand-1
226
198 (87.6)
116 (51.3)
109 (48.2)
103 (45.6)
102 (45.1)
–
–
0
0
0
96 (42.5)
83
44 (53.0)
35 (42.2)
35 (42.2)
30 (36.1)
24 (28.9)
–
–
4 ( 4.8)
–
–
19 (22.9)
0
Bogor (isk)-1
Thailand-1
Hiroshima-1
Bangladesh-1
103
52 (50.5)
52 (50.5)
48 (46.6)
27 (26.2)
7 ( 6.8)
0
7
16 (15.5)
5
0
3 ( 2.9)
Okinawa
Bangladesh-1
204
190 (93.1)
67 (32.8)
57 (27.9)
52 (25.5)
2 ( 1.0)
1
1
18 ( 8.8)
–
–
0
Malaysia
Bangladesh-1
761
747 (98.2)
204 (26.8)
172 (22.6)
116 (15.2)
2 ( 0.3)
0
2
16 ( 2.1)
5
0
Thailand-1
Bangladesh-1
161
74 (46.0)
38 (23.6)
38 (23.6)
37 (23.0)
38 (23.6)
–
–
0
0
0
38 (23.6)
Bangladesh-1 Bangladesh-1
246
164 (66.7)
164 (66.7)
153 (62.2)
146 (59.4)
134 (54.5)
5
0
0
0
0
114 (46.3)
89
39 (43.8)
39 (43.8)
36 (40.5)
35 (39.3)
20 (22.5)
–
–
6 ( 6.7)
–
–
26 (30.3)
11 ( 6.7)
0
11
45 (27.4)
5
0
10 ( 6.1)
1 ( 0.5)
0
Bogor (isk)-1
Bangladesh-1
Hiroshima-1
Bogor( isk)
164
101 (61.6)
101 (61.6)
92 (56.1)
62 (37.8)
Okinawa
Bogor( isk)
201
177 (88.1)
80 (39.8)
68 (33.8)
61 (30.4)
0
0
0
37 (18.4)
–
–
Malaysia
Bogor( isk)
254
182 (71.7)
72 (28.4)
46 (18.1)
30 (11.8)
0
0
0
16 ( 6.3)
–
–
Thailand-1
Bogor( isk)
250
193 (77.2)
52 (20.8)
52 (20.8)
50 (20.0)
45 (18.0)
–
–
5 ( 2.0)
–
–
49 (19.6)
Bangladesh-1 Bogor( isk)
506
454 (89.7)
283 (55.9)
174 (34.4)
160 (31.6)
150 (29.6)
–
–
4 ( 0.8)
–
–
149 (29.5)
83
76 (91.6)
17 (20.5)
17 (20.5)
16 (19.3)
16 (19.3)
5
0
0
0
0
16 (19.3)
Bogor (isk)-1
Bogor( isk)
0
–, not examined
Table 8. Developmental capacity of the hybrid among 5 Asian populations of the F. limnocharis complex and the controls in the second
crossing experiments
Female
Parents
Male
No. of
Eggs
Hiroshima-2
Hiroshima-2
185
No. of normally
cleaved eggs
(%)
168 (90.8)
No. of normal
tail-bud embryos
(%)
165 (89.2)
No. of normally
hatched tadpoles
(%)
163 (88.1)
No. of normally
No. of 30-day-old tadpoles
feeding tadpoles
Normal
Underdeveloped
(%)
(%)
(%)
130 (70.3)
129 (69.7)
1 ( 0.5)
Thailand-2
Hiroshima-2
294
259 (88.1)
81 (27.6)
63 (21.4)
46 (15.7)
3 ( 1.0)
41 (14.0)
Bangladesh-2
Hiroshima-2
523
490 (93.7)
472 (90.3)
465 (88.9)
457 (87.4)
0
402 (76.9)
23 ( 4.4)
Bangladesh-3
Hiroshima-2
364
334 (91.8)
311 (85.4)
293 (80.5)
275 (75.6)
3 ( 0.8)
230 (63.2)
Hiroshima-2
Cianjur (lim)
192
157 (81.8)
155 (80.7)
150 (78.1)
147 (76.6)
147 (76.6)
Thailand-2
Cianjur (lim)
290
232 (80.0)
44 (15.2)
23 ( 7.9)
9 ( 3.1)
8 ( 2.8)
Bangladesh-2
Cianjur (lim)
344
119 (34.6)
92 (26.7)
88 (25.6)
85 (24.7)
Bangladesh-3
Cianjur (lim)
372
290 (78.0)
274 (73.7)
261 (70.2)
254 (68.3)
Thailand-2
Thailand-2
258
228 (88.4)
143 (55.4)
120 (46.5)
Bangladesh-2
Thailand-2
508
495 (97.4)
487 (95.9)
400 (78.7)
Banglades-2
Thailand-2
365
355 (97.3)
339 (92.9)
313 (85.8)
288 (78.9)
285 (78.1)
Hiroshima-2
Bangladesh-2
216
211 (97.7)
197 (91.2)
185 (85.7)
184 (85.2)
Thailand-2
Bangladesh-2
341
303 (88.9)
35 (10.3)
18 ( 5.3)
12 ( 3.5)
11 ( 3.2)
1 ( 0.3)
11 ( 3.2)
Bangladesh-2
Bangladesh-2
580
567 (97.8)
457 (78.8)
402 (69.3)
398 (68.6)
274 (47.2)
86 (14.8)
272 (46.9)
Bangladesh-3
Bangladesh-2
402
381 (94.8)
277 (68.9)
268 (66.7)
235 (58.5)
215 (53.5)
11 ( 2.7)
209 (52.0)
Hiroshima-2
Cianjur (isk)
126
125 (99.2)
122 (96.8)
118 (93.7)
117 (92.9)
Thailand-2
Cianjur (isk)
264
259 (98.1)
168 (63.6)
81 (30.7)
33 (12.5)
33 (12.5)
0
30 (11.4)
Bangladesh-2
Cianjur (isk)
393
379 (96.4)
354 (90.1)
310 (78.9)
35 ( 8.9)
26 ( 6.6)
3 ( 0.8)
26 ( 6.6)
Bangladesh-3
Cianjur (isk)
395
380 (96.2)
367 (92.9)
287 (72.7)
7 ( 1.8)
5 ( 1.3)
1 ( 0.3)
1 ( 0.3)
41 (14.0)
0
0
1 ( 0.3)
No. of normally
metamorphosed frogs
(%)
120 (64.9)
69 (19.0)
146 (76.0)
8 ( 2.8)
76 (22.1)
9 ( 2.6)
1 ( 0.3)
217 (58.3)
50 (13.4)
84 (32.6)
58 (22.5)
8 ( 3.1)
47 (18.2)
388 (76.4)
386 (76.0)
1 ( 0.2)
375 (73.8)
3 ( 0.8)
274 (75.1)
0
0
160 (74.1)
97 (77.0)
0
0
370
T. H. Djong et al.
In the first crossing experiments using females from
Thailand, the ova appeared to be of low quality, and the
developmental capacities of the controls and hybrids were
reduced. For the crossing experiments involving females
from the Thailand population, the eggs cleaved normally
with frequencies ranging from 9.1–77.2% (x– =42.7%),
became normal tail-bud embryos with frequencies ranging
from 9.1–49.1% (x– =29.6%), hatched normally with frequencies ranging from 9.1–49.1% (x– =29.3%), became normally
feeding tadpoles with frequencies ranging from 9.1–47.4%
(x– =27.6%), became normal 30-day-old tadpoles with frequencies ranging from 0–26.9% (x– =12%) or underdeveloped with frequencies ranging from 0–47.4% (x– =15.6%),
and finally metamorphosed normally with frequencies ranging from 0.8–40.9% (x– =22.9%).
The survival curves of the controls and the hybrids
between several Asian populations and topotypic F.
limnocharis and F. iskandari are shown in Fig. 4. The
crossing experiments revealed that hybrids between male
topotypic F. limnocharis and females from the Hiroshima,
Fig. 4. Survival curves of hybrids between several Asian populations and topotypic Fejervarya limnocharis and F. iskandari from Java (Indonesia) and the controls. (A) First cross between females from several Asian populations and male topotypic F. limnocharis. (B) Second cross
between females from several Asian populations and male topotypic F. limnocharis. (C) First cross between females from several Asian populations and male topotypic F. iskandari and the control. (D) Second cross between females from several Asian populations and male topotypic
F. iskandari and the control. (E) First cross between female topotypic F. iskandari and males from several Asian populations and the control.
Speciation in Asian Fejervarya Complex
Malaysia and Okinawa populations became normal tadpoles
with the capacity for metamorphosis at certain ratios. In contrast, almost all the hybrids between females from Thailand
(first cross, Thonphaphum) and Bangladesh (first and second crosses) and males from topotypic F. limnocharis populations became underdeveloped (Fig. 5) and died before or
just after metamorphosis. A small number of the hybrids
between female F. iskandari and male F. limnocharis
became normal tadpoles and underwent metamorphosis.
371
These normal tadpoles were found to be triploid (3n) in their
chromosome number (Fig. 4A, B, Table 7). In the second
crossing experiments using a female from Pathumthani of
Thailand (a local sample different from that used for the first
crossing experiments) and males from the Hiroshima, Bangladesh and topotypic F. iskandari populations, the hybrids
were able to undergo metamorphosis (Table 8).
The crossing experiments between females from the
Hiroshima (first and second crosses), Okinawa and Kuala
Fig. 5. Thirty-day-old tadpoles of hybrids among the Fejervarya limnocharis complex from Asia and the controls. (A) Thailand♀×topotypic F.
limnocharis♂. (B) Bangladesh♀×topotypic F. limnocharis♂. (C) Topotypic F. iskandari♀×topotypic F. limnocharis♂. (D) Thailand control.
(E) Bangladesh control. (F) Topotypic F. iskandari control. Scale bar=1.0 cm.
Fig. 6. Thirty-day-old tadpoles of hybrids among the Fejervarya limnocharis complex from Asia and the controls. (A) Hiroshima ♀×topotypic
F. iskandari♂. (B) Malaysia♀×topotypic F. iskandari♂. (C) Okinawa♀×topotypic F. iskandari♂. (D) Hiroshima control. (E) Malaysia control.
(F) Topotypic F. iskandari control. Scale bar=1.0 cm.
372
T. H. Djong et al.
Lumpur (Malaysia) populations and male F. iskandari from
Java revealed that all the 30-day-old tadpoles became
underdeveloped (Fig. 6) and died before metamorphosis,
except in the first crosses between a Hiroshima female and
a male F. iskandari, in which some tadpoles developed normally and reached metamorphosis. Reciprocal crosses
between female F. iskandari and males of the Hiroshima
and Kuala Lumpur (Malaysia) populations also resulted in a
large number of underdeveloped tadpoles and only a few
normal tadpoles (Fig. 4C–E). By checking the chromosome
number, the normal hybrids were found to be triploid (3n),
while the underdeveloped hybrids were diploid (2n) (Table
7). The normal hybrids obtained from the crosses between
female F. iskandari and males from the Thailand and Bangladesh populations and the reciprocal crosses were able to
reach metamorphosis.
DISCUSSION
The simple definition of a species is a group of interbreeding natural populations that are reproductively isolated
from other such groups (Mayr, 1969). Species are also
viewed as genetic systems delimited by isolation mechanisms and genetically based traits that prevent gene
exchange (Dobzhansky, 1937; Mayr, 1942). Reproductive
isolation is essential for morphological, ecological or genetic
divergence between sympatric or allopatric groups (Turelli et
al., 2001). Genetic divergence results in reproductive isolation that reflects further accumulation of genetic differentiation at many loci over a long period of time (Ferguson, 2002;
Sites and Marshall, 2003, 2004). Wu and Hollocher (1998)
argued that if large numbers of genes are responsible for
reproductive isolation between taxa and there are gradual
changes at all these loci over time, there should be a correlation between genetic divergence and the degree of reproductive isolation. Such correlations between reproductive
isolation and genetic divergence have been demonstrated
in Drosophila (Coyne and Orr, 1989), the salamander
Desmognathus ochrophaeus (Tilley et al., 1990), the salamander Plethodon (Highton, 1989, 1990) and some anuran
species (Sasa et al., 1998).
Genetic divergence is measured by calculating the
genetic distance that represents the degree of dissimilarity
between the genetic compositions of taxa, and therefore
appears to be an ideal systematic tool (Ayala, 1975; Bullini,
1983). According to Thorpe (1982), allopatric populations
with genetic identities above 0.85 should be considered to be
conspecific, while those with identities below 0.85 should be
considered to be different species. Highton (1989) used Nei’s
distance of 0.15 to delimit species within the salamanders
(Plethodon) of North America. Rafinski and Arntzen (1987)
found that the genetic distances among Triturus species in
the Palearctic region were 0.41–2.00. Nishioka and Sumida
(1990) reported that the genetic distances between populations and species of 15 anuran species were 0.030–0.241
and 0.301–1.715, respectively. Skibinski et al. (1993)
reported that the genetic distances among different vertebrate species were above 0.147. Beerli et al. (1994) showed
that the genetic distances between the water frog Rana
cerigensis and nine other species in Europe were 0.27–0.95.
Sumida and Nishioka (1996) reported that the genetic distances among different populations of 55 amphibian species
were 0.007–0.205. Veith et al. (2002) found that the mean
genetic distance among populations of Rana temporaria
from Germany was 0.121, while distances between R. temporaria and Rana pyrenaica, Rana iberica and Rana macrocnemis were 0.181– 0.667. Zangari et al. (2006) found that
the genetic distances between Discoglossus species in the
Mediterranean area were 0.17–1.04. Sasa et al. (1998)
found positive correlations between the degree of divergence
(Nei’s (1972) genetic distance) and the degree of postzygotic
isolation in anurans, and a lower threshold of genetic distance (0.3) for the evolution of hybrid inviability. Sites and
Marshall (2003, 2004) assumed that reproductive isolation is
based on divergence across many loci scattered throughout
the genome, and that allozyme loci diverge in an approximately clock-like manner, so as to correlate with, and serve
as a signature for, the emergence of reproductive isolation.
Thus, it may be roughly concluded that allopatric populations
with genetic distances above 0.30 can be considered to be
different species, and that this value can be considered to
represent the threshold of genetic distance for the emergence of reproductive isolation such as hybrid inviability.
Previous studies on the genetic divergence of the F. limnocharis complex have been carried out by several research
groups. Nishioka and Sumida (1990) reported that the mean
genetic distance among seven populations in western Japan
was 0.077. The Iriomote population was differentiated from
the Hiroshima, Okinawa and Taiwan populations with a
mean genetic distance of 0.320. Toda et al. (1997) further
demonstrated that the mean genetic distance between the
southern Ryukyu (including Iriomote Island) and East Asian
populations was 0.59. These differences indicate that the
absolute divergence values obtained from allozyme analyses are not always consistent with each other due to differences in the number of loci scored, interpretations of electromorphs observed in the genotypes, etc. Toda et al.
(1998b) reported that F. limnocharis in Taiwan had diverged
into two groups, the eastern and western groups, with
genetic distances of 0.129–0.305. Based on allozyme data,
Toda et al. (1998a) found a cryptic species from Java with
a genetic distance of 0.458 from the syntopic F. limnocharis,
and Veith et al. (2001) described it as a new species, F.
iskandari, with a genetic distance of 0.316 from the syntopic
F. limnocharis. Maeda and Matsui (1999) regarded the
Sakishima Island populations of F. limnocharis, including the
Ishigaki and Iriomote Islands in Japan, as a distinct species
on the basis of morphological, acoustic and genetic differences. Several investigators have reported conspicuous
morphological and genetic divergence of the Sakishima
Island populations from other populations of this species
(Kuramoto, 1979; Nishioka and Sumida, 1990; Sumida et
al., 2002; Toda et al., 1997; Toda, 1999). On the basis of
morphological observations, allozyme data, molecular data
and crossing experiments, Sumida et al. (2002) suggested
that it is reasonable to regard the Sakishima Island populations as a subspecies of F. limnocharis.
In the present study based on allozyme data (genetic
distances and NJ tree topology) and crossing experiments,
the F. limnocharis complex from various localities was
shown to have largely diverged into two groups, the smalltype and large-type groups. The mean genetic distance
among the small-type populations was 0.833, while those
Speciation in Asian Fejervarya Complex
between the small-type and large-type populations were
1.160–1.408 (Table 9). In the NJ tree, the small-type group
formed one cluster with a BT value of 72.7%, and the mean
Fst value among the small-type populations was 0.843. In
fact, crossing experiments have already revealed that smalltype populations are reproductively isolated from large-type
populations by complete hybrid inviability at the embryonic
stage (Sumida et al., unpublished data). This finding clearly
indicates that populations belonging to the small- and largetype groups represent different species. Since the large-type
group includes F. limnocharis sensu stricto and F. iskandari,
Table 9.
373
the nomenclature of populations of the small-type group
requires consideration. In the small-type group of the F.
limnocharis complex, the Bangladesh (BAU(S)) population
was more closely related to the Sri Lanka population than
the Indian and Thailand populations. Specifically, the
genetic distances were 0.181 between the Bangladesh
(BAU(S)) and Sri Lanka populations, 0.588 between the
Indian and Sri Lanka populations, 0.526 between the Indian
and Bangladesh (BAU(S)) populations and 1.104–1.310
between the Thailand population and the populations of
India, Sri Lanka and Bangladesh (Table 6). Based on mito-
Summary of the mean genetic distance (range) among the F. limnocharis complex from Asian countries
Large type
Species (Population)
F. iskandari
Indonesia
Small type
F. limnocahris complex
Indonesia
Malaysia
Thailand
Bangladesh
Outgroup
F. cancrivora
Japan
Taiwan
India
Sri Lanka
Bangladesh
Thailand
F. iskandari (Indonesia)
0.024
(0.012–0.032)
F. limnocharis (Indonesia)
0.661
(0.628–0.720)
0.060
(0.046–0.076)
F. limnocharis (Malaysia)
0.543
(0.470–0.614)
0.451
(0.410–0.526)
0.144
F. limnocharis (Thailand: Large type)
0.373
(0.285–0.431)
0.431
(0.250–0.572)
0.370
(0.287–0.469)
0.115
(0.056–0.197)
F. limnocharis (Bangladesh: Large type)
0.271
(0.249–0.291)
0.534
(0.509–0.579)
0.353
(0.311–0.393)
0.308
(0.220–0.408)
–
F. limnocharis (Japan, Taiwan)
0.387
(0.300–0.455)
0.480
(0.365–0.638)
0.430
(0.353–0.488)
0.309
(0.191–0.501)
0.238
(0.188–0.291)
0.112
(0.027–0.290)
F. limnocharis (India, Sri Lanka,
Bangladesh,Thailand: Small type)
1.160
(0.709–1.495)
1.408
(1.137–1.652)
1.323
(1.055–1.466)
1.126
(0.919–1.393)
1.287
(0.864–1.633)
1.350
(0.980–1.730)
0.833
(0.181– 1.310)
F. cancrivora (Indonesia)
1.171
(1.162–1.238)
1.342
(1.241–1.432)
1.174
(1.102–1.246)
0.952
(0.846–1.062)
1.018
1.026
(0.910–1.294)
1.564
(1.407–1.694)
Indonesia
–
Parentheses show the range values.
Fig. 7. Reproductive-isolation mechanisms found in various combinations among the Fejervarya limnocharis complex from Asia. a)Two different populations were used: the first cross involved the Thonphaphum population and the second involved the Pathumthani population. Normal
*
viability in the second cross.
374
T. H. Djong et al.
chondrial 16S rRNA gene sequence data, Sumida et al.
(unpublished data) also found that the Bangladesh BAU(S),
Sri Lanka and Fejervarya syhadrensis populations from
India made one cluster in a NJ tree, and were distinctly
diverged from the Indian and Thailand (Pilok) populations.
These data possibly imply that our specimens from the Sri
Lanka and Bangladesh (BAU(S)) populations belong to F.
syhadrensis, and that the Indian and Thailand (Pilok) populations are a different species. The Thailand (Pilok) and
Indian populations may possibly be a new species.
In the present study, we used F. limnocharis and F.
iskandari from the type localities on Java, Indonesia. The
mean genetic distance between these two species, 0.661
(range, 0.628–0.720), was the highest among the large-type
group, whereas that among F. iskandari populations from
Java was 0.024 (range, 0.012–0.032) and that among F.
limnocharis populations from Java was 0.060 (range, 0.046–
0.076) (Table 9). Furthermore, the results of the crossing
experiments between these species revealed that they are
isolated by complete hybrid inviability at the tadpole stage
(Fig. 7). This result corroborates the conclusions of Toda et
al. (1998a) and Veith et al. (2001) that these sympatric species, although very difficult to distinguish from each other
morphologically, can be clearly distinguished by allozyme
analyses. In the present study, F. iskandari and F. limnocharis had diagnostic alleles at 11 loci of 27 presumptive
loci examined. Toda et al. (1998a) found diagnostic alleles
at 10 of 25 loci examined, and Veith et al. (2001) found diagnostic alleles at six of 15 presumptive loci analyzed. We
found both species in each of three populations from Java,
although the percentage compositions of F. limnocharis
were very low (range, 1.7–26.1%; mean, 15.9%). Moreover,
the genetic variability of F. limnocharis was lower than that
of F. iskandari (mean values, H=6.3, P=19.7 and A=1.24 for
F. limnocharis and H=9.6, P=46.9 and A=1.64 for F.
iskandari).
Based on the present allozyme data, the large-type
group could be divided into two subgroups. One subgroup
consisted of the Bangladesh, F. iskandari and JapaneseTaiwanese populations, while the other subgroup consisted
of the Thailand, Malaysia and F. limnocharis populations.
These results were contradictory with those of the crossing
experiments that showed no reproductive isolation as hybrid
inviability among the Japanese-Taiwanese, Malaysia and F.
limnocharis populations or among the F. iskandari, Thailand
and Bangladesh populations. Based on mitochondrial 16S
rRNA gene sequence data, Sumida et al. (unpublished data)
found that the F. iskandari, Thailand and Bangladesh populations comprise one cluster that is diverged from another
cluster consisting of the F. limnocharis, Malaysia and
Japanese-Taiwanese populations. In addition, the allozyme
NJ tree showed no confidence in either of the two large-type
subgroups. Therefore, we designated one subgroup including F. iskandari and the Thailand and Bangladesh populations as the F. iskandari subgroup, and the other subgroup
including F. limnocharis and the Malaysia and JapaneseTaiwanese populations as the F. limnocharis subgroup. The
mean genetic distances were 0.271–0.373 among the F.
iskandari subgroup and 0.430–0.480 among the F.
limnocharis subgroup (Table 9), and no reproductive-isolation mechanisms existed among each subgroup until the
metamorphosis stage. If we apply the genetic distance criteria for allopatric populations defined by Thorpe (1982),
Highton (1989) and Skibinski et al. (1993) and the correlation between the genetic distance and reproductive isolation
(Sasa, 1998; Sites and Marshall, 2003, 2004), as already
mentioned in the second paragraph of the Discussion, the F.
limnocharis complex in the Thailand and Bangladesh populations may be regarded as a subspecies or another species
of F. iskandari from the type locality of Java, and the F.
limnocharis complex from the Malaysia and JapaneseTaiwanese populations may be regarded as a subspecies or
another species of F. limnocharis from the type locality of
Java, since the mean genetic distances among the F.
limnocharis complex from these five countries ranged from
0.238 to 0.661 in the present study. This statement is supported by mean genetic distances of 0.144, 0.112 and 0.115
among the Malaysian populations, Japanese-Taiwanese
populations and Thailand populations, respectively (Table
9). Besides, the Fst values among subgroup F. iskandari and
subgroup F. limnocharis were 0.604 and 0.818, respectively,
and higher than those among F. iskandari from Java and F.
limnocharis from the Java and Thailand populations (0.128,
0.356 and 0.419, respectively). Further studies investigating
the fertility or spermatogenesis of mature hybrids, detailed
morphological characteristics and molecular divergences
will be required to elucidate the exact taxonomic status of
the F. limnocharis complex from each country in Asia.
ACKNOWLEDGMENTS
We are grateful to Professor Yong Hoi Sen and Mr. Daicus M.
Belabut of University of Malaya, Drs. T. Fujii and H. Hasegawa of
the University of the Ryukyus, and Dr. M. Katsuren of the Okinawa
Prefectural Institute of Health and Environment, for their kindness in
making F. limnocharis samples from Kuala Lumpur, Okinawa and
Iriomote, respectively, available for this study. The first author,
Djong Hon Tjong, is grateful to Mr. Yaya Hernawan SP from
Soekarno-Hatta Fish Quarantine, Centre for Fish Quarantine, Ministry of Marine Affairs and Fisheries, Indonesia, who permitted the
transport of frog samples from Indonesia (No. 03545.TI210.2010.IV.2005). This work was supported by Technological and
Professional Skills Development Sector Project (TPSDP) ADB Loan
No. 1792-INO, Directorate General of Higher Education, Department of Education, Indonesia (to Djong Hon Tjong), and by a Grantin-Aid for Scientific Research (C) (No. 17570082) from the Ministry
of Education, Culture, Sports, Science and Technology, Japan (to
M. Sumida).
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(Received May 30, 2006 / Accepted November 27, 2006)